To understand the molecular mechanisms underlying neurotransmitter release and its modulation, we are identifying proteins including Snapin, syntaphilin, and syntabulin that control the trafficking, targeting, and assembly of presynaptic release machinery at nerve terminals. We are further analyzing the functional roles of these proteins using molecular, biochemical, cell biological, and electrophysiological analysis (collaborating effort with other labs). The continued application of these approaches, in combination with a greater focus on cellular siRNA knockdown and genetic knockout studies, will allow us to better understand the regulatory pathways that govern the synaptic vesicle exocytosis and regulate synaptic strength. Syntabulin is one of our major focuses during the fiscal year 2004. The process of neurotransmitter release involves a series of protein-protein interactions between the membranes of synaptic vesicles and presynaptic terminals. Syntaxin-1 is a key component of the membrane fusion machinery at the presynaptic plasma membrane. Presynaptic proteins involved in synaptic vesicle exocytosis are synthesized in the soma of neurons, incorporate into transport cargo vesicles, and undergo proper trafficking to the synaptic terminal. However, little is known regarding the mechanisms by which cargo organelles containing vesicle release machinery physically associate with their microtubule-based transport motors. In an effort to characterize syntaphilin isoforms, we have identified a gene encoding a novel syntaxin-binding protein named syntabulin. Syntabulin attaches syntaxin-containing vesicles to microtubules and migrates with syntaxin within the processes of hippocampal neurons. Knockdown of syntabulin expression with targeted siRNA or interference of the syntabulin-syntaxin interaction inhibit attachment of syntaxin-cargo vesicles to microtubules and reduce syntaxin-1 distribution in the neuronal processes. Furthermore, molecular motor protein kinesin I KHC binds to syntabulin and associates with syntabulin-linked syntaxin vesicles in vivo. These findings suggest that syntabulin acts as a linker molecule that attaches syntaxin-cargo vesicles to motor kinesin I, enabling the transport of syntaxin-1 to neuronal processes. Our findings provide new clues to the cellular mechanisms that are likely to underlie the specific trafficking of synaptic cargoes.